Self-assembling nanofibrous bacteriophage microgels as sprayable antimicrobials targeting multidrug-resistant bacteria

Nanofilamentous bacteriophages (bacterial viruses) are biofunctional, self-propagating, and monodisperse natural building blocks for virus-built materials. Minifying phage-built materials to microscale offers the promise of expanding the range function for these biomaterials to sprays and colloidal bioassays/biosensors. Here, we crosslink half a million self-organized phages as the sole structural component to construct each soft microgel. Through an in-house developed, biologics-friendly, high-throughput template method, over 35,000 phage-built microgels are produced from every square centimetre of a peelable microporous film template, constituting a 13-billion phage community. The phage-exclusive microgels exhibit a self-organized, highly-aligned nanofibrous texture and tunable auto-fluorescence. Further preservation of antimicrobial activity was achieved by making hybrid protein-phage microgels. When loaded with potent virulent phages, these microgels effectively reduce heavy loads of multidrug-resistant Escherichia coli O157:H7 on food products, leading to up to 6 logs reduction in 9 hours and rendering food contaminant free.

This is very well written article that reports the fabrication of densely packed phage microgels with a porous fibrous structure. The article is clear and detailed, providing ample information to understand the manipulations performed. The results are well presented and conclusions well substantiated in general by the results. The results achieved are interesting and appear to be novel (although some clarifications are required -see my comments below) and the fibrous structures obtained are well characterized and very appealing. I can foresee several potential applications of these microgels in the biomedical and environmental fields. I recommend publication of this article, after the authors address the few questions and comments below: -The authors report a glutaraldehyde and an EDC-based methods for crosslinking phages, and that they then mold into honeycomb structures. While the molding strategy may be novel for phage hydrogels, phages have been crosslinked previously with the same crosslinkers. For instance, the Belcher group at MIT has demonstrated the fabrication of GA-crosslinked hydrogels (and aerogels), and EDC-crosslinked thin films (see a few published articles below). It appears like the authors are achieving a high packing density for phages, but can the authors more directly comment on the novelty of their methods and / or the differences in phage density / packing / structure compared with the previously reported crosslinked phage gels from literature? Highly adjustable 3D nano-architectures and chemistries via assembled 1D biological templates, Nanoscale 2019 Assembly of a Bacteriophage-Based Template for the Organization of Materials into Nanoporous Networks, Adv Mater 2014 Versatile Three-Dimensional Virus-Based Template for Dye-Sensitized Solar Cells with Improved Electron Transport and Light Harvesting, ACS Nano 2013 -Could the authors clarify why the crosslinks between phages do not affect the bioactivity of the phages? What are the important features of the phage for antibacterial activity? Based on antibacterial assays, it appears like BSA is a crucial component of the gel that allows for preserving phage activity -however, the abstract and introduction elute to phage gels without specifically mentioning BSA. If BSA is essential for phage activity, then I think that it should be explicitly mentioned from the beginning of the article that the microgels are composed of phage AND BSA. -Page 6, lines 20-21: "A single M13 phage exhibits abundant amine and carboxyl groups (5,400 and 10,800, respectively) on its protein coat…" -I assume that the authors are referring to only the reactive subset of amine and carboxylic groups from on the coat proteins of the M13 phage. I believe that many more would be present, but all may not be reactive. This is not a major comment, but worth clarification by the authors. How were these numbers estimated (from the 2700 copies of the coat protein)? -Page 7, lines 1-2: "…less than 12 hrs to gel completely… " What is meant by "complete" gelation? -It is not immediately clear to me what the purpose of the honeycomb molding described on page 7 is. Why was this type of mold select over other molding techniques or mold shapes for the gels? -In BSA/phage gels, are the same advantages of densely packing phages preserved? Are there tradeoffs to consider between phage-only and BSA-phage hydrogels? Can BSA interfere with some of the potential applications of the phage microgels? -Based on the FTIR results shown in Figure 3, would the authors be able to calculate a density or ratio of crosslinked amine groups (and carboxylic acid groups) relative to all available groups on the phage? -The authors rationalize using EDC as alternative crosslinker because of the autofluorescence observed with GA, and the need for gel that do not autofluoresce for "some application scenarios, for example certain biosensing applications that rely on fluorescence to detect target analytes". Could the authors be more specific? How would fluorescent probes eventually be incorporated in the phage hydrogels for these applications?
Reviewer #3 (Remarks to the Author): The most noteworthy results are the fabrication and potential use of bacteriophagecontaining microgels which will add to the existing delivery systems for phages. The methodology is sound, based on established techniques for gelation by crosslinking and characterisation including antimicrobial tests. The manuscript contains sufficient details in the methods for the work to be reproduced. The quality of the work certainly meets the expected standards in the field.
The work is not totally original as the related concept of using filamentous phages to form hydrogels has been explored and reported by the same research team (references 15-17). Hybrid hydrogels of phage M13 and BSA without cross-linkers have already been reported (reference 16), using glutaraldehyde as a widely used protein cross-linker (ref 17). The use of EDC as a crosslinker instead of glutaraldehyde in this paper is thus useful. As the authors stated, application of the established fabrication approach of forming the hexagonal template casting (ref 31,32) is not novel, also was previously reported by the same group, but the novelty lies in the application of the method to prepare phage microgels which is the theme of this paper.
The work does not provide data to support some of the conclusions, eg, on the claims of a high throughput method, as the results focused on the preparation of the microgels and their characterization, instead of study to confirm the high throughput nature of the method. Also, the authors claimed that 'the microgels also protected against desiccation' but there are no long-term stability results to support that claim.                 Revised manuscript, page 1 "Here, we crosslink half a million self-organized phages as the sole structural component to construct each soft microgel"  Revised manuscript, page 1 "Nanofilamentous bacteriophages (bacterial viruses) are biofunctional, self-propagating, and monodisperse natural building blocks for virus-built materials.
Minifying phage-built materials to microscale offers the promise of expanding the range function for these biomaterials to sprays and colloidal bioassays/biosensors." "phage-built microgels" "an in-house developed, biologic-friendly, high-throughput template method"  "self-organized, highly-aligned nanofibrous texture and tunable autofluorescence"  "When loaded with potent virulent phages, these microgels effectively reduce heavy loads of multidrug-resistant Escherichia coli O157:H7 on food products, leading to up to 6 logs reduction in 9 hours and rendering food contaminant free."  "We demonstrate that two crosslinkers can each effectively assist the gelation of phage nanofilaments through different crosslinking reactions, leading to vastly different fluorescence profiles. We further show that the phage nanofilaments in these virus-exclusive microgels selfassemble into an orderly, highly aligned nanofibrous structure that serve as a high-load delivery vehicle for protein and strong virulent phages to control multidrug-resistant E. coli O157:H7 in food products."   Introduction:"We further show that the phage nanofilaments in these virus-exclusive microgels self-assemble into an orderly, highly aligned nanofibrous structure that serve as a high-load vehicle for protein and strong virulent phages to control multidrug-resistant E. coli O157:H7 in food products."      "In summary, we obtained over 3.5×10 4 phage microgels from every square centimeter of our template. Every film we made was over 5 cm 2 , allowing for the production of 175,000 phage microgels in a single day. In addition, more than 10 films can be applied to produce microgels simultaneously, demonstrating the high-throughput ability of this method. Each microgel contains more than 3.8×10 5 phage particles, constituting a phage community of 10 10 in total."   